Sex-Biased Expression of Genes Allocated in the Autosomal Chromosomes: Blood LC-MS/MS Protein Profiling in Healthy Subjects

Background Sex and gender have a large impact in human health and disease prediction. According to genomic/genetics, men differ from women by a limited number of genes in Y chromosome, while the phenotypes of the 2 sexes differ markedly. Methods In this study, serum samples from six healthy Bahraini men and women were analyzed by liquid chromatography–mass spectrometry (LC-MS/MS). Bioinformatics databases and tools were used for protein/peptide (PPs) identification and gene localization. The PPs that differed significantly (p < 0.05, ANOVA) in abundance with a fold change (FC) of ≥1.5 were identified. Results Revealed 20 PPs, 11 were upregulated in women with very high FC (up to 8 folds), and 9 were upregulated in men but with much lower FC. The PPs are encoded by genes located in autosomal chromosomes, indicative of sex-biased gene expression. The only PP related to sex, the sex hormone-binding globulin, was upregulated in women. The remaining PPs were involved in immunity, lipid metabolism, gene expression, connective tissue, and others, with some overlap in function. Conclusions The upregulated PPs in men or women are mostly reflecting the functon or risk/protection provided by the PPs to the specific sex, e.g., Apo-B100 of LDLC. Finally, the basis of sex-biased gene expression and sex phenotypic differences needs further investigation.


Introduction
Biological and physiological diferences exist between men and women [1]. Te conventional molecular distinctions between the two sexes are mainly the genomic makeup and transcriptomic typing [2,3], while the proteomic diferences are persuaded by epigenetics, alternative splicing, posttranslation modifcations, as well as others [4]. However, the infuence of sex hormones on gene expression should not be overlooked. A new and expanded central dogma composed of OMICs platform, constituted of genome, transcriptome, and proteome, is needed for better utilization of the human genome data. Although the human genome is expected to constitute between 20,000 and 25,000 genes [5], more than 2 million proteins and peptides (PPs) are detected by proteomics [6]. In addition to their normal physiological roles, PPs are central players in disease etiology, pathology, and complications as well as they are utilized as diagnostic and prognostic biomarkers [7].
In the era of personalized and precision medicine, sex (biological determinants of men and women) and gender (environmental and cultural determinants) stand early during the diversifcation of people into categories and subcategories down to the level of individuals. Terefore, an initial step towards the personalization of medicine would be setting the molecular diferences between men and women. Te inherent susceptibility of men and women to disease is known to vary considerably, e.g., men are more prone to infections, coronary heart disease (CHD), while women are more prone to autoimmune and infammatory diseases [8][9][10][11]. Moreover, it is well known that women have more vigorous innate immunity [9] and humoral immunity to antigenic challenges [12], which accelerate pathogen clearance but can lead to an increased frequency of immunological disorders such as autoimmune or infammatory diseases [10,13]. Moreover, it has been observed that sex/ gender afects the responses to vaccination and its outcome [14]. Finally, women's pregnancy is another determinant for immune and infammatory responses [15].
Proteomics is an up-and-coming discipline emerged from the Human Genome Project and became an indispensable platform for a better understanding of genomic and transcriptomic data [16]. Liquid chromatography-mass spectrometry (LC-MS/MS) is the major tool that has revolutionized the proteomic analysis [17]. Complimented with bioinformatics, the proteomics resolved the issue of protein/ peptide detection, quantitation, and together with sequence identifcation, they facilitate protein structure and function prediction and their possible roles in health and disease. Moreover, the proteomic analysis is superior to the other biochemical and antibody-based approaches in the ability to detect with high accuracy and sensitivity the small diferences in PPs in study materials [18]. Lastly, the used techniques and software are regularly upgraded for the generation and analysis of larger data [19].
Usually, studies of the diferences between men and women are focused on physiological events, susceptibility to a specifc disorder, drug response/interaction, or variations of biochemical markers [20]. Te sex-specifc physiological events or disorders, e.g., pregnancy and lactation or breast and prostatic tumors, were organ-specifc with diferent markers and susceptibility factors [21]. However, only very few studies have focused on the diferences between the two sexes in the health status [22]. Te data of the present study are part of a larger study of the blood proteomic changes in T2DM, where several proteins were identifed to be differentially expressed in T2DM, mostly were upregulated [6]; however, the infuence of gender on T2DM and other disorders cannot be corrected for by the traditional proteomic statistical analysis. Terefore, in this study, we compared the serum protein profle of healthy men and women who were included in the main study.

Study Subjects.
A subset of samples was obtained from 6 healthy subjects, 3 males/3 females, aged 41, 42, and 47 years (age and sex matched), selected from a larger number of Bahraini volunteers involved in the study of T2DM biomarkers, between September 2014 and February 2015 (Table 1). Te main inclusion criteria were original Bahraini national, nondiabetic, apparently healthy, male or female, and willing to participate in the study. Exclusion criteria included younger (<40 years) and relatively older (>50 years) ages, acute or chronic disorders, prolonged treatment, e.g., antibiotics and immune suppressive drugs. Informed consent was obtained from each study subject before blood collection. Te study design followed the Helsinki Declaration terms. Te study was approved by the Research and Ethics Committees of the Arabian Gulf University (AGU) and Salmaniya Medical Complex Hospital (SMC), Manama, Bahrain.

Protein Profling
Using LC-MS/MS. Te protein profle analysis was done by LC-MS/MS as previously described [23,24], in brief;

Serum Preparation -Protein Depletion.
Pierce albumin/IgG removal kit (Termo Scientifc, U.S.A.) was used to remove the major subclasses of gamma globulin (IgG) and human serum albumin (HSA) from serum as the most abundant serum proteins, in order to detect the least abundant ones (depleted serum). Te unprocessed serum (nondepleted serum) was also used. Tereafter, the exact quantity of proteins in serum was determined in order to calculate the amount of sample needed for analysis.

In-Solution Protein
Digestion. Te serum samples, both nondepleted and depleted, were diluted 1: 1 with 0.1% RapiGest ™ SF (Waters, UK). Tereafter, the sera were pooled into 2 sets of samples, 3 healthy males and 3 healthy females. Te total load for each pooled sample for analysis was 100 μg of protein/peptide (PPs) in a fnal volume of 25 μl. Ten, the PPs in the pooled samples were denatured in a Termo mixer R (Eppendorf, Hamburg, Germany) at a speed of 8 × g at 80°C for 15 min. In the end, the PPs were digested by trypsin at a 1 : 50 ratio (Promega Corporation, Madison, WI, USA). Te PPs digests were then analyzed using Synapt G2 MS (Waters, Manchester, UK).

Protein Identifcation. Te 1-dimensional Nano
Acquity liquid chromatography coupled with tandem mass spectrometry on a Synapt G2 instrument (Waters, Manchester, UK) was used for label-free quantitative expression protein profling. For ESI mass spectrometry analyses, the instrument settings were optimized on the MassLynx tune page. A protein digest of 3-μg was loaded on the column, and the samples were spiked with yeast alcohol dehydrogenase (ADH, P00330) as an internal standard to digest to give 200 fmol per injection for absolute quantitation. An Acquity sample manager was used for the injection of the analyzed samples.
Te samples were analyzed in duplicate runs, and data were acquired using the MassLynx program (version. 4.1, SCN870; Waters) operated in resolution and positive polarity modes. Te acquired MS data were background subtracted, smoothed, and deisotoped at the medium threshold. Progenesis QI V2.0 (QIfp) for proteomics (Nonlinear Dynamics/Waters, UK) was used for automated data processing and database searching. Te generated peptide masses were searched against in UniProt speciesspecifc protein sequence database using the Progenesis QI V2.0 (QIfp) for proteomics for protein identifcation and quantifcation (Nonlinear Dynamics/Waters, UK).

Data Analysis and Informatics
2.4.1. Proteomics Data. Progenesis QI V2.0/TransOmics Informatics (Waters Scientifc, Manchester, UK) software was used to process and search the data using the principle of search algorithm as previously described [24,25]. Te data were fltered to show only statistically signifcant diferences (p < 0.05, ANOVA) coupled with a change in proteins' abundance by 1.5 fold or more (Maximum Fold Change-MFC ≥1.5). Additionally, the absolute quantifcation was performed using ADH as an internal standard to give an absolute amount of each identifed protein/peptide.

Abundance of Upregulated PPs in Healthy Women and
Men. Figure 1 shows the diferentially expressed PPs, the PPs which were upregulated in women (A), and the ones which were upregulated in men (B). Te diferences in the abundance of PPs in the serum between the two sexes were rated by the maximum fold change (MFC). Te PPs, which were increased by more than 3 folds, i.e., MFC >3, were mostly upregulated in women. Tese PPs in decreasing order were apolipoprotein C-II (8.6 folds), homeobox protein Hox-D13 (6.0 folds), apolipoprotein A-II (4.0 folds), isoform 4 of coiled-coil domain-containing protein 17 (3.6 folds), and alpha-1-acid glycoprotein 2 (3.4 folds). However, in men, only the isoform 4 of fbronectin was increased by more than 3 folds compared with women (MFC 3.02). For the fold changes and p-values, see Table 2 and Figure 1.  Table 2.    (Figures 3(a) and 3(b)). Te expression profles of the identifed PPs in nondepleted serum (Figure 2(a)) and depleted serum (Figure 3(a)) were strongly diferentiated between women and men using correspondence analysis. Te hierarchical cluster analysis of the expression profles of the identifed PPs in nondepleted sera is shown in (Figure 2(b)), and that of the PPs in depleted sera is shown in Figure 3(b), which both diferentiated between men and women. Of these PPs, the ones which were expressed predominantly in the liver are 4 PPs, in lymphocytes and bone marrow cells were 3 PPs and 1 PP in the CNS (corpus callosum), while only 2 PPs were expressed predominantly by females' genital organs. Te classes of PPs upregulated in both women and men were the lipid metabolism and immunity classes, and in women, the 2 classes included the following PPs: apolipoprotein A-II, apolipoprotein C-II and CD5 antigen-like, and Ig mu chain C. Te acute phase proteins (alpha-1-acid glycoprotein 1 and alpha-1-acid glycoprotein 2) and signal transduction (multiple PDZ domain protein) classes of PPs were upregulated exclusively in women. Furthermore, 2 PPs are involved in gene expression; the homeobox protein Hox-D13 and isoform 4 of coiled-coil domain-containing protein 17, and one PP (integrin beta-2) classifed as connective tissue and another as transporter (sex hormone-binding globulin) PP, were upregulated also in women.   Genetics Research

Proteins/Peptides (PPs) Upregulated in Healthy Men.
Te PPs upregulated in men compared to women were 9 PPs (Figure 1(b)), 7 were identifed in nondepleted serum while 2 were identifed in depleted serum. Using the correspondence analysis, the expression profles of the former (Figure 2(a)) and the latter (Figure 3(a)) PPs strongly diferentiated men from women. As shown in Figures (2(b) and 3(b)), the hierarchical cluster analysis of the expression profles of the 7 PPs in the nondepleted and that of the 2 PPs in depleted sera, respectively, clearly distinguished between men and women. Tree of the upregulated PPs in men, the complement factor H, Ig gamma-1 chain C region, and Ig gamma-2 chain C region, belong to the immunity class of PPs. Two PPs were classifed under each of the lipid metabolism (Apo-B100 and isoform SH-iPLA2 of 85/88 kDa calcium-independent phospholipase A2) and connective tissue (isoform 4 of fbronectin and vitronectin) classes. One PP, the eukaryotic translation initiation factor 4E type 3 (fragment), is involved in gene expression, specifcally translation. However, one PP, the isoform 2 of putative golgin subfamily A member 2B, is likely to be allocated to Golgi apparatus, but its class is unknown.

Ingenuity Pathway Analysis (IPA).
Te ingenuity pathway analysis (IPA) is used for meaningful interpretation of gene expression data using prior biological knowledge. For networking of the identifed PPs between each other and other PPs sharing the same function, and for casual and functional relation to disease and immunity, the 20 PPs were subjected to IPA, using an all-in-one, web-based software application, from QIAGEN Bioinformatics. Only 14 PPs (7 were upregulated in men and 7 in women) ( Table 3) were mapped in the IPA database together with another 18 PPs not identifed in this study (Figure 4). Tese PPs were found to be implicated in several networks. Te gene sources of these proteins were highlighted in grey in Figure 4. Te cellular localization of some of these PPs included the plasma membrane, cytoplasm, nucleus, and extracellular space (Table 3).

Discussion
Te fact that men and women biologically, morphologically, and physiologically are diferent [26] is inspiring to study the nature and magnitude of this diference at the molecular level. It is well known that genomically and genetically both sexes possess the same chromosomes and genes except for the sex chromosome (XY), namely the Y chromosome genes, e.g., SRY gene [27]. In this study, proteomic analysis revealed marked diferences in the levels of 20 PPs, none of which is encoded by genes located in the sex chromosomes, controversial to what was observed elsewhere [28]. Furthermore, the PPs upregulation was more evident in women, confrming a recent report showing signifcant sexual dimorphism in protein abundance between twin pairs of the opposite sex [28], although men have the unique chromosome Y. Te observation that all identifed PPs are expressed by genes located in the autosomal chromosomes is supported by a previous study reporting that the sex diferences in part are due to diferences in the expression of genes not in sex chromosomes [29]. Furthermore, 3 PPs known to be regulators of gene expression were diferentially expressed in this study, supporting the sex-biased expression independent of the sex chromosomes. Two transcription factors, homeobox protein Hox-D13 and isoform 4 of coiled-coil domain-containing protein 17 (CCDC17), were upregulated in women, while a translational factor, eukaryotic translation initiation factor 4E (eIF4E) type 3 (Fragment), was upregulated in men. Te Hox gene family products are known to be transcriptional factors involved in female reproductive system development and function [30]. However, the other 2 PPs are not known to have any sexspecifc role. Te only PPs in this study that was directly related to sex were the sex hormone-binding globulin (SHBG), which was upregulated in women. One study showed that SHBG mRNA is strongly correlated with serum SHBG protein level, with higher levels of mRNA and protein in women than in men [31], in line with the present study.
Of the markedly diferentially expressed PPs, 11 were upregulated in women and 9 in men; however, the diferences in abundance were more marked in the PPs upregulated in women. In this study, the experimental system was set to select only markedly diferentially expressed PPs; however, in biological systems, function and homoeostasis depend on a fne balance of molecules, i.e., not necessarily the quantity [32,33]. Noticeably, was the upregulation of Apo AII and Apo CII in women, and that of Apo B100 in men. Te former two were the predominant Apo proteins in chylomicron and VLDL, which are not involved directly in atherosclerosis and coronary heart disease (CHD), while (Te image was generated using J-express pro V1.1 software program (java.sun.com)). (b) Unsupervised hierarchical cluster analysis of the expression profles of depleted serum samples using 5 proteins that difer signifcantly (P < 0.05, analysis of variance; maximum fold change ≥1.5) between men (red) and women (blue). Te dendrogram was generated using the Bray-Curtis correlation distance metric and an average linkage clustering method from the J-express pro V1.1 software program (java.sun.com). Note. One PP (IGHG1) was upregulated in both nondepleted ( Figure 2) and depleted sera.

Genetics Research
Apo B100, the major protein in LDL, is the carrier of pathological cholesterol [34,35]. It is well known that men are more susceptible to CHD compared to women of the same age before menopause [8], which is consistent with the present fnding. Te sex-based immunological dimorphism was known a long time ago [36]; however, detailed analysis showing the abundance of immunoglobulins (Ig) chains at the transcriptome or proteome level was rarely investigated. In this study, the upregulation of Ig mu chain C region (IgM heavy chain) in women and that of Ig gamma-1 and 2 chain regions (IgG heavy chains) and complement factor H (the complement alternative pathway) in men probably refects the diference between the two sexes in response to antigenic challenges. In line with our fndings are Saudi and Iranian studies, both showed higher levels of IgM in healthy women compared with men and higher IgG in men compared with women in the Saudi but not in the Iranian study [37,38]. Studies of sex diferences in immune responses following infection/vaccination showed that women across all age groups were able to mount higher immune responses to infections and vaccines than men [14,39]. In contrast, women compared to men are at much higher risk for development of autoimmune diseases, e.g., SLE [40].
To the best of our knowledge, the diferences in the blood levels of the acute phase proteins, alpha-1-acid glycoprotein-1 and 2 (AGP1 and 2), also known as orosomucoid, between men and women were not reported before. In this study, both PPs were upregulated in women. Te associations of AGPs with rheumatoid arthritis [41] and other autoimmune diseases, e.g., Grave's disease [42], together with the high prevalence of these disorders in women [11], are strongly supporting our fndings. For signal transduction PPs, the Genetics Research multiple PDZ domain protein, which is known to have the highest expression level in the corpus callosum (brain), was the only PP identifed in this study. It is involved in the control of cell polarity and signal transduction [43,44], and it is also known to be associated with several and diverse pathologies [45], thus, its upregulation in women is worth further investigation. In this study, the upregulation of the PPs in men was not as high as in women in terms of abundance (fold change). Te upregulation of the 2 connective tissue (CT) proteins, fbronectin and vitronectin in men, is not unexpected since men and women have diferent musculoskeletal system properties [46]. Te sex diference in the levels of both PPs was not reported before; however, both proteins were shown to be upregulated in T2DM [6,47]. Te diferences in CT proteins between the 2 sexes have clinical implications; e.g., the incidence of anterior cruciate ligament injury is almost 10 times higher in women compared with men performing the same activity [48], an observation that supports our fndings.
Te last PP that was upregulated in men is the isoform 2 of putative golgin subfamily A, member 2B, one of the Golgi apparatus proteins, which is probably encoded by a pseudogene (https://www.genecards.org/cgi-bin/carddisp.pl?gene= GOLGA2P5). However, no single article was published about this PP in the PubMed database. Tis and other PPs listed in Table 2 are not discussed here but are worth further investigation.
As a summary of the above, we further explored the functional characteristics and relatedness of the 20 PPs with disease and other immune-mediated disorders using ingenuity pathway analysis (IPA). Te principle of the IPA was explained previously [49]. Only 14 of the 20 PPs were mapped in the IPA database and were found to be implicated in multiple signaling networks including cell-to-cell signaling and interaction, lipid metabolism, and small molecule Figure 4: Pathway analysis of network signaling of 32 proteins represented in the ingenuity pathway analysis database included 14 PPs identifed in this study (grey shading). Only thirty-two (32) of the 100 proteins were mapped in the IPA database and were implicated in the pathway analysis of multiple signaling networks including cell-to-cell signaling and interaction, cell death and survival, cellular movement, and immune cell trafcking (the image was generated using ingenuity pathway analysis program (IPA version 49:309) https://qiagen.force. com).
biochemistry. Te functional annotation of these proteins with others as transporters, transmembrane receptors, and catalysts is referred to in Table 3; moreover, these networks are in connections with diferent drug agents (data not shown).
Of the limitations of this study is the small sample size, and lack of validations of the LC-MS/MS results with other methods, although results were previously validated by testing 3 PPs including fbronectin in 80 plasma samples by ELISA [47].
In conclusion, 20 PPs were found to be diferentially expressed in healthy men and women, 11 upregulated in women with high fold change, while 9 were upregulated in men with lower fold change compared to women. Although diferentiating between the two sexes, none of the identifed PPs is encoded by genes allocated to sex chromosomes. Te frequently obvious links between sex and PPs upregulation, and the physiological and pathological roles of the latter are validation for this proteomic data; however, further investigation is needed. Te diferences between men and women at the molecular level are crucial for a better understanding of sex and gender-related disorders which is the frst step towards precision/personalized medicine. Although the biological diferences between men and women were thoroughly studied, to the best of our knowledge, this is the frst study in humans' proteomics for the same purpose.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
In this study, the procedures involving human participants were done in accordance with the ethical standards of the Research Committees of the Arabian Gulf University and SMC (Manama, Bahrain) and with the 1964 Helsinki Declaration and its later amendments. An informed consent was obtained from each subject.

Consent
Te patient consented for publication.

Conflicts of Interest
All authors declare that they have no conficts of interest.